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Cordyceps militaris is widely cultivated as a functional food and traditional medicine in China and Southeast Asia. In this study, C. militaris hybrid strains were isolated from different parts of hybrid fruiting body cultivated through mating-based sexual reproduction of ascospores. We investigated the effect of strains, solid-state substrates and light condition on production of yield and contents of bioactive compounds (cordycepin and adenosine) using HPLC in fruiting bodies. The results indicated that yield and production of bioactive compounds exist distinct differences on strain separated part, solid-substrates and light conditions. The middle strain obtained the highest yield on wheat substrate. The yield was higher on wheat substrate, the concentration of cordycepin and adenosine in fruiting bodies were higher on rice substrate. The red light is beneficial to high yield, and white light is conducive to the accumulation of cordycepin in fruiting bodies, the light condition has little effect on the adenosine content.